Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine, diethylnitrosamine and methyl methanesulphonate, on liver regenerating after partial hepatectomy. II. Alkylation of DNA and inhibition of DNA replication.

Experiments were carried out to determine whether replication of alkylated DNA could be involved in the initiation of hepatocellular carcinoma which results from a single administration of dimethylnitrosamine (DMN) given after partial hepatectomy. The incidence of tumours is higher when DMN is given during the wave of DNA synthesis induced by the operation than when given in the early prereplicative stage. Therefore the alkylation of DNA in the regenerating liver by DMN given at these times and the effect of DMN on DNA synthesis were investigated. The extent, duration and pattern of alkylation of DNA, including the formation of 0-6-methylguanine, were similar whether DMN was given in the early pre-replicative stage (6 h after the operation) or during the period of DNA synthesis (at 24 h). DMN given a 6 h very greatly reduced the wave of DNA replication which would otherwise have ensued. When given at 24 h, by which time DNA synthesis was already taking place, DMN reduced the rate of incorporation of (-3H)thymidine after 1-2 h delay. However, in neither case was DNA synthesis reduced to the level occurring in normal intact liver. Treatment with diethylnitrosamine (DEN) at 6 h or at 24 h had a similar effect to DMN on the wave of DNA replication induced by partial hepatectomy. Methyl methanesulphonate (MMS given in the early pre-replicative stage delayed the wave of DNA synthesis by about 8 h, but when it did take place the extent of synthesis was as great as in untreated animals. When given during the period of DNA replication, MMS rapidly reduced the rate of synthesis. As in the case of the nitrosamines, synthesis was not reduced to the level occuring in normal intact animals. The difference from the nitrosamines lies in the nature of the alkylated bases formed in DNA. The fact that a single treatment with DMN induces cancer in partially hepatectomised animals but not in intact adult animals is not considered to be due to a gross difference in the nature of the alkylation of DNA. The experiments described support the concept that replication of DNA containing bases which are likely to mispair during replication may be necessary to 'fix' the lesion and thus cause a permanent inheritable change in the genetic material.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. IV. Effect on methylase-mediated methylation of DNA.

The possibility that carcinogens may affect methylase-mediated methylation of replicating DNA was investigated. A system eminently suitable for this purpose is liver regenerating after partial hepatectomy, as one injection of dimethylnitrosamine (DMN) given during the ensuing period of increased DNA synthesis induces hepatocellular carcinoma. Methylation of DNA by DNA methylase normally occurs only in proportion to DNA synthesis. Therefore simultaneous measurements were made of synthesis (incorporation of [14C]adenine into DNA adenine, or of d[5-3H]cytidine into DNA cytosine), and of methylation (incorporation of [methyl-3H]methionine into 5-methylcytosine of DNA) in liver regenerating after partial hepatectomy. After treatment with DMN, the ratio of methylation: synthesis remained within the normal range. Methyl methanesulphonate (MMS), a compound which damages DNA in regenerating liver in a similar but not identical way to DMN and which does not induce tumors in liver even when given after partial hepatectomy, caused an increase in methylation in relation to synthesis. These experiments therefore do not support the view that altered DNA methylase activity is involved in carcinogenesis.     

Nitrosamine-induced carcinogenesis. The alkylation of nucleic acids of the rat by N-methyl-N-nitrosourea, dimethylnitrosamine, dimethyl sulphate and methyl methanesulphonate

1. N[(14)C]-Methyl-N-nitrosourea, [(14)C]dimethylnitrosamine, [(14)C]dimethyl sulphate and [(14)C]methyl methanesulphonate were injected into rats, and nucleic acids were isolated from several organs after various time-intervals. Radioactivity was detected in DNA and RNA, partly in major base components and partly as the methylated base, 7-methylguanine. 2. No 7-methylguanine was detected in liver DNA from normal untreated rats. 3. The specific radioactivity of 7-methylguanine isolated from DNA prepared from rats treated with [(14)C]dimethylnitrosamine was virtually the same as that of the dimethylnitrosamine injected. 4. The degree of methylation of RNA and DNA produced in various organs by each compound was determined, and expressed as a percentage of guanine residues converted into 7-methylguanine. With dimethylnitrosamine both nucleic acids were considerably more highly methylated in the liver (RNA, about 1% of guanine residues methylated; DNA, about 0.6% of guanine residues methylated) than in the other organs. Kidney nucleic acids were methylated to about one-tenth of the extent of those in the liver, lung showed slightly lower values and the other organs only very low values. N-Methyl-N-nitrosourea methylated nucleic acids to about the same extent in all the organs studied, the amount being about the same as that in the kidney after treatment with dimethylnitrosamine. In each case the RNA was more highly methylated than the DNA. Methyl methanesulphonate methylated the nucleic acids in several organs to about the same extent as N-methyl-N-nitrosourea, but the DNA was more highly methylated than the RNA. Dimethyl sulphate, even in toxic doses, gave considerably less methylation than N-methyl-N-nitrosourea in all the organs studied, the greatest methylation being in the brain. 5. The rate of removal of 7-methylguanine from DNA of kidneys from rats treated with dimethylnitrosamine was compared with the rate after treatment of rats with methyl methanesulphonate. No striking difference was found. 6. The results are discussed in connexion with the organ distribution of tumours induced by the compounds under study and in relation to the possible importance of alkylation of cellular components for the induction of cancer.     

Effect of a single treatment with the alkylating carcinogens dimethylnitrosamine and methyl methanesulphonate on liver regenerating after partial hepatectomy. III. Effect on DNA synthesis in vivo and on DNA polymerase activity assayed in vitro.

A single treatment with dimethylnitrosamine (DMN) but not with methyl methanesulphonate (MMS) induces liver cell carcinoma if given during the period of restorative hyperplasia following partial hepatectomy, a higher incidence of tumours being induced if the carcinogen is given during the period of DNA synthesis (24 h after the operation) than if given early in the prereplicative stage (at 6 h). To study the effect of treatment with DMN and with MMS on the regenerating liver, DNA replication was measured in vivo in partially hepatectomised animals treated with the methylating agents, and DNA polymerase activity was assayed in vitro.     

Reaction of carcinogen dimethylnitrosamine with proteins and with thiol compounds in the intact animal

1. [¹⁴C]Dimethylnitrosamine was administered to rats, and the formation of free and protein-bound S-methylcysteine was investigated. 2. Very little S-methylcysteine was found in liver acid-soluble fraction, in liver protein or in urine. 3. The methylhistidines formed in liver protein were found to have largely disappeared by 6 days after injection of dimethylnitrosamine. 4. The significance of these results in the carcinogenic action of dimethylnitrosamine is discussed.     

Effect of treatment in vivo with N,N-dimethylnitrosamine or methyl methanesulphonate on the cytoplasmic DNA polymerase of regenerating rat liver.

A 10-16 fold increase in rat liver cytoplasmic DNA polymerase (DNA polymerase-alpha) was observed by 24 hrs after two thirds partial hepatectomy. Injection of either N,N-dimethylnitrosamine (DMN) or methyl methanesulphonate (MMS) At 6 or 12 hrs after partial hepatectomy completely inhibited this increased production of polymerase, but when given at 20 hours they had less effect. Neither compound altered the molecular size distribution of the enzyme. These data indicate that the lowered levels of DNA polymerase-alpha could play a major role in the reduction in DNA synthesis which occurs after carcinogen treatment.     

Repair replication in human cells studied by sodium iodide isopycnic centrifugation of DNA in a fixed-angle rotor

A method for purification of ethynyl steroids from biological fluids has been developed using silver-sulfoethyl cellulose column chromatography. Ethynyl steroids were applied in methanol, and the firm binding allowed the columns to be washed with methanol, ethyl acetate, or diethyl ether to remove endogenous materials. Nonethynylated steroids did not bind to silver-sulfoethyl cellulose. Release of ethynylated steroids was achieved with a saturated NaCl/methanol solution. Dehydration or de-ethynylation of ethynyl estradiol, ethynodiol diacetate, and ethynodiol was not observed. The utility of this technique for purification of ethynyl steroid metabolites from the urine of a beagle metabolizing norethynodrel was demonstrated.     

A comparative study of hepatic DNA repair, DNA replication and hepatotoxicity in the CD-1 mouse following multiple administrations of dimethylnitrosamine

The objective of this study was to quantify hepatic DNA repair and DNA replication following multiple administrations of dimethylnitrosamine (DMN) and to determine if these events were correlated with hepatotoxicity. Male CD-1 mice, 50-100 days old, were dosed daily, p.o., with DMN in water at dose levels of 2, 4, 7 and 10 mg/kg for 2 weeks. After 2, 7 and 14 days of dosing, hepatocytes were isolated by an in situ perfusion procedure, incubated in the presence of [3H] thymidine, and fixed. Unscheduled as well as scheduled DNA synthesis were assessed by quantitative autoradiography. Unscheduled DNA synthesis (UDS) represents DNA repair while scheduled DNA synthesis (S phase) represents DNA replication. In addition, the animals' serum was examined for enzymes which indicate hepatic toxicity. After 1, 7 and 14 days of dosing, animals were orbital-bled and the serum was analyzed for serum glutamic pyruvic transaminase (SGPT), serum glutamic oxalacetic transaminase (SGOT), alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT). No morbidity or mortality was observed at dose levels of 2 and 4 mg/kg, but all animals receiving 7 and 10 mg/kg died after 4-6 days of dosing. GGT or AP were not elevated at any dose level or at any time point examined. At 4 mg/kg only a slight increase (less than or equal to 2 X) in the concentration of SGOT and SGPT was observed but a sharp increase (greater than 20 X) in replicative DNA synthesis was seen. The 2 mg/kg dose level of DMN did not increase replicative DNA synthesis and SGOT and SGPT were not elevated above control values at any time point following dosing at 2 mg/kg. A weakly positive DNA repair response was observed for dose levels of 4, 7 and 10 mg/kg DMN after two consecutive days of dosing. No DNA repair was observed after either 7 or 14 days of dosing at the 2 and 4 mg/kg/day levels. These results indicate that hepatic toxicity is associated with the induction of replicative DNA synthesis (S phase) but not with the induction of DNA repair. The results also confirm and extend a previous study (Doolittle et al., 1987b) indicating that a significant elevation in hepatic DNA replication is induced by hepatocarcinogens after multiple administrations of dose levels which do not alter hepatic DNA replication after a single administration. This finding indicates that the utility of the in vivo-in vitro hepatocyte assay may be enhanced by using a multi-dose protocol.     

DNA replication in eucaryotic nuclei. Evidence suggesting a specific model of replication

Preimplantation-stage mouse embryos suspended in dimethyl sulfoxide (DMSO) have been used as a model to study details of the response of a simple multicellular system to freezing and thawing. Rapid freezing to ?196 °C kills the embryos unless they have first been cooled very slowly to at least below ?50 °C. The survival of both 2-cell and 8-cell embryos has been found to depend as critically on the rate at which the frozen embryos were thawed as on the rate at which they were first frozen. The damaging consequences of thawing frozen embryos too rapidly have been shown to occur between ?70 and ?20 °C. Finally, the survival of embryos as a function of the time in DMSO prior to freezing and thawing has been compared with their volume changes as a function of time in DMSO. This comparison leads to the tentative conclusion that dimethyl sulfoxide need not permeate the embryos to protect them against freezing damage. Overall, the embryos' response to freezing and thawing is qualitatively similar to that displayed by many other cell types.     

Repair processes and the response of dividing and non-dividing cells to methyl methanesulphonate and dimethyl sulphate.

A method for producing a viable non-dividing population of Chinese hamster V79 cells in suspension is described and the characteristics of the population outlined. The stationary population is more sensitive to methylating agents than a similar but exponentially growing population, the increased sensitivity arising from the loss of the shoulder from the survival curve. The extent of reaction of the agent with cellular macromolecules is similar in both cases. The repair capabilities of the two populations was examined. Non-semiconservative DNA repair synthesis occurs whether the cells are in a growth or no-growth condition when insulted. Repair of single-strand breaks, which arise following methylation, also proceeds up to the size of the replicon. The relationship of this stationary population to other no-growth conditions and its utility as a model for carcinogenesis studies is discussed.     

Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei

Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery.     

Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs

We demonstrate that cell-free extracts prepared from activated eggs of X. laevis by a method similar to that of Lohka and Masui initiate and complete semiconservative DNA replication of sperm nuclei and plasmid DNA. The efficiency of replication is comparable to that in the intact egg. Under optimal conditions 70%-100% of nuclei, and up to 38% of naked DNA molecules replicate completely. Genuine initiation of replication occurs rather than elongation of preformed primers or priming of irreversibly denatured templates. Rereplication of templates is observed under certain conditions. In addition to replicating DNA, these extracts also assemble nucleus-like structures from naked DNA.     

Repair of DNA damaged by methyl methanesulfonate in bacteriophage T4.

A highly purified preparation of T4 endonuclease V does not degrade DNA alkylated with methyl methanesulfonate, and the methyl methanesulfonate sensitivity of T4 wild type and x mutant is not affected by the v mutation. Thus, T4 endonuclease V, the v gene product, does not seem to be involved in a repair or an abortive repair of methyl methanesulfonate-damaged T4 DNA. The x and y genes of T4 and the polA and the uvrD genes of Escherichia coli are concerned with the repair of methyl methanesulfonate-induced damages in T4 DNA. Since effects of the polA and the x or y mutations are additive, it is supposed that there are at least two pathways for the repair of methyl meth-anesulfonate-damaged T4 DNA, one controlled by the x and the y genes and the other in which E. coli DNA olymerase I is involved. The partial suppression of the x gene mutation at high temerature was also demonstrated.     

DNA strand breakage caused by dichlorvos, methyl methanesulphonate and iodoacetamide in Escherichia coli and cultured Chinese hamster cells

The DNA damaging properties of dichlorvos (2,2 dichlorovinyl dimethyl phosphate), methyl methanesulphonate (MMS) and iodoacetamide (IAA) have been studied, using alkaline sucrose sedimentation. In a strain of E. coli deficient in DNA polymerase I (polA) both dichlorvos and MMS caused random strand breakage, MMS being about twice as efficient as dichlorvos on a molar basis. In pol⁺ bacteria, DNA strand breaks or alkali labile bonds were detected following treatment with roughly five-fold higher concentrations of MMS but at similar high concentrations of dichlorvos there was an all or none breakdown of DNA molecules to fragments of very low molecular weight which correlated well with lethality.DNA synthesized after treatment of pol⁺ and polA bacteria with MMS was of low molecular weight, indicating the presence of discontinuities. With dichlorvos, the effect was much smaller.Apparent all-or-none DNA breakdown was also found when the polA strain of E. coli was treated with low concentrations of iodoacetamide, an agent that does not detectably alkylate DNA. At higher concentrations the breakdown was suppressed and random strand breakage occurred instea. These effects did not occurr with pol⁺ bacteria and correlated well with the greater sensitivity to iodoacetamide of the polA strain in survival experiments. We suggest that the major DNA damage resulting from treatment with iodoacetamide and dichlorvos arises indirectly through alkylation of other cellular constituents and consequent uncontrolled nuclease attack on the DNA. Discontinuities in newly synthesized DNA and mutagenesis following dichlorvos treatment, however, presumably result from direct alkylation of DNA.Strand breakage caused by dichlorvos and MMS in Chinese hamster cells tended to correlate with the extent to which these agents alkylate DNA, but survivval tended to correlate with the alkylation of protein.     

Repair replication in replicating and non-replicating DNA after irradiation with UV light.

Ultraviolet light induces more pyrimidine dimers and more repair replication in DNA that replicates within 2 to 3 hr of irradiation than in DNA that does not replicate during this period. This difference may be due to special conformational changes in DNA and chromatin that might be associated with semiconservative DNA replication.     

Regulation of DNA replication in the nuclei of the slime mold Physarum polycephalum. Transplantation of nuclei by plasmodial coalescence

Nuclei in G2 phase of the slime mold Physarum polycephalum, when transplanted, by plasmodial coalescence, into an S-phase plasmodium, failed to start another round of DNA synthesis. In the reciprocal combination, S-phase nuclei in a G2-phase host continued DNA synthesis for several hours without appreciable decrease in rate. It is suggested that the beginning of DNA replication is determined by an event, either during or shortly after mitosis, which renders the chromosomes structurally competent for DNA replication.     

Stability of deoxyribonucleic acid methylated in the intact animal by administration of dimethylnitrosamine. Rate of breakdown in vivo and in vitro at different dosages

1. The effect of administration of various dosages of dimethylnitrosamine on the extent of methylation of liver and kidney nucleic acids in the intact rat was studied. Methylation of liver nucleic acids was linearly related to the dosage, but decreasing the dose produced relatively less lowering of the extent of alkylation of kidney nucleic acids. 2. The rates of disappearance of 7-methylguanine from DNA during the 2 days after administration of dimethylnitrosamine in the intact animal and on incubation under simulated physiological conditions in vitro were compared. At a high dosage this rate was greater in vivo than in vitro. At a low dosage the small difference between the two rates was not thought to be sufficient evidence for existence of a specific enzymic excision of the abnormal base.